Factor VII composition having a substantially homogenous isoelectric point

ABSTRACT

The present invention relates to a factor VII composition having a substantially homogeneous isoelectric point and to a method for formulating such a composition. The present invention also relates to the therapeutic use of a factor VII composition having a substantially homogeneous isoelectric point.

This application is a divisional of U.S. application Ser. No.14/896,763, filed Dec. 8, 2015, which is a U.S. National Stage ofPCT/EP2014/062051, filed Jun. 10, 2014, which claims priority to FrenchPatent Application No. 13/55403, filed Jun. 11, 2013, all of which arehereby incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a factor VII composition having asubstantially homogeneous isoelectric point and to a method forformulating such a composition. The present invention also relates tothe therapeutic use of a factor VII composition having a substantiallyhomogeneous isoelectric point.

TECHNOLOGICAL BACKGROUND

The Factor VII (FVII) is a glycoprotein depending on vitamin K, which isinvolved in the extrinsic route of blood coagulation. In its activatedform (FVIIa), the factor VIIa is involved in the coagulation process byforming a complex with the tissue factor (TF) and by activating thefactor X and the factor IX into factor Xa and IXa, respectively. SoFVIIa has the capability of triggering coagulation of blood when thecascade of reactions leading to blood coagulation is interrupted ordeficient, for example in the absence of factor VII or IX. This is whythe factor VIIa has been used for a long time as a drug for treatingcertain disorders of blood coagulation expressed by bleeding, andnotably for treating patients having factor VIII deficiency (hemophiliaof type A) or factor IX deficiency (hemophilia of type B) and havinginhibitors against these factors, for treating patients havingcongenital factor VII deficiency, or as a product for preventinghemorrhages which may occur during surgical operations.

The factor VII is secreted in the form of a single peptide chain of 406amino acids, with a molecular weight of about 50 kDa, which is cut atthe Arg₁₅₂-Ile₁₅₃ (Arginine 152-Isoleucine 153) bond during itsactivation into FVIIa. The Factor Vila resulting from this thereforeconsists of a light chain of 152 amino acids, with a molecular weight ofabout 20 kDa. and of a heavy chain of 254 amino acids, with a molecularweight of about 30 kDa, bound together through a single disulfide bridge(Cys₁₃₅-Cys₂₆₂).

Four distinct structural domains may be identified in the sequence ofthe factor VII: an N-terminal γ-carboxylic domain (Gla domain), two“epidermal growth factor (EGF)-like”: domains as well as a serineprotease domain.

The plasma Factor VIIa moreover includes several post-translationalmodifications among which: γ-carboxylation of the first ten glutamicacids of its sequence, partial hydroxylation of the aspartic acid 63,O-glycosylation of the serines 52 and 60 (Ser₅₂ and Ser₆₀) andN-glycosylation of the asparagines 145 and 322 (Asn₁₄₅ and Asn₃₂₂)(Fenaille F. et al., Mass spectrometric characterization of N- andO-glycans of plasma-derived coagulation factor VII. Glycoconj. J. 25.9(2008): 827-42).

The biological activity of the factor VII thus strongly depends on thenature and on the proportion of the oligosaccharide structures which areattached to the protein, and which may affect many aspects of thetherapeutic efficiency such as for example, solubility, resistance toproteolytic attacks, thermal inactivation, immunogenicity, half-life,bioactivity, bioavailability and stability of the factor VII.

It appears that these post-translational modifications, which often varyfrom one plasma factor VII molecule to another, lead to someheterogeneity of the molecules present in the pharmaceuticalcompositions of factor VII. Such heterogeneity notably has the drawbackof complicating the steps for formulating the factor VII with view tothe preparation of pharmaceutical compositions intended for treatingpatients. Indeed, it is frequent that a significant proportion of thepurified factor VII molecules precipitate during the formulation step:the result of this is a loss of active raw material, as well as thepresence of the factor VII having become inactive into pharmaceuticalcompositions intended for therapeutic treatment.

Because of the numerous drawbacks of the use of human plasma (risks ofviral contamination, difficulty in purification, supply . . . ) as asource of pharmaceutical products, it is now preferred to produce thefactor VII in recombinant or transgenic systems. However, insofar thatglycosylation is a complex post-translational modification whichdirectly depends on the cell system used, large scale production ofproteins in heterologous cells often leads to the production ofpolypeptides having an identical primary structure, but having variableoligosaccharide structures.

For example, this is the case of NovoSeven®, a drug which has beenauthorized on the European market since 1996 and authorized on theAmerican market in 1999, produced by the Danish firm NovoNordisk, theactive ingredient of which is eptacog alfa (human recombinant activatedcoagulation Factor VII produced by genetic engineering from BKH kidneycells of newborn hamsters).

This is also the case, of recombinant transgenic FVII described inapplication EP 2 037 955 A, filed on May 31, 2007 by LFBBiotechnologies.

It thus appears that for these recombinant factors VII, theheterogeneity of the post-translational modifications borne by themolecules of Factor VII perturb the formulation step and contribute toreducing the stability and/or the specific activity of the finalpharmaceutical products, by promoting the presence of precipitatedfactor VII molecules. The result of this is many difficulties formanaging to prepare pharmaceutically acceptable compositions having auniform and predetermined clinical efficiency.

Therefore there exists an increasing need for factor VII compositionshaving improved chemical and physical stability at room temperature andcapable of facilitating and improving the yield of the formulation stepsaiming at preparing pharmaceutical compositions intended for thetreatment of patients affected by hemophilia with inhibitors orcongenital factor VII deficiency.

SUMMARY OF THE INVENTION

The applicant has surprisingly discovered that factor VII compositions,for which the factor VII molecules have a substantially homogeneousisoelectric point facilitate the formulation step at an optimum pH,preferably at an optimum pH of 6.0±0.2, of pharmaceutical compositionswhile avoiding precipitation of the factor VII.

The present invention therefore relates to a factor VII composition inwhich the factor VII molecules have a substantially homogeneousisoelectric point.

In a particular embodiment, the present invention relates to a factorVII composition in which, among all the N-glycan forms of the factor VIIof the composition, at least 60% of said N-glycan forms are monochargedand among all the factor VII molecules of the composition, at least 80%of said molecules have γ-carboxylation on 9 residues of glutamic acid.

In a particular embodiment, at least 65%, preferably at least 70% orpreferably at least 80% of the N-glycan forms of the factor VII of thecomposition are monocharged.

In a particular embodiment, at least 85%, preferably from 85% to 100% orpreferably from 90% to 100%, or preferably from 95% to 100% of thefactor VII molecules of the composition have γ-carboxylation on 9residues of glutamic acid.

In a particular embodiment, the γ-carboxylation level of the factor VIIcomposition of the invention on the residue of glutamic acid 35 (Glu₃₅relatively to the sequence of the plasma factor VII) is less than 20%,or preferably less than 15%, or still preferably less than 10%,preferably less than 5%.

In a particular embodiment, the present invention relates to a factorVII composition, for which at least 60% of the N-glycan forms of thefactor VII of the composition are monosialylated complexes.

In a particular embodiment, at least 10%, preferably at least 15%, orpreferably at least 20%, or preferably at least 25% of the N-glycanforms of the factor VII of the composition are high Mannose/hybrids.

In a particular embodiment at least 90%, preferably at least 95% of thefactor VII molecules of the composition have an isoelectric pointcomprised in a pH unit interval of less than 1.2.

In a particular embodiment, at least 50/o %, or preferably at least 55%or preferably 60% of the factor VII molecules of the composition have anisoelectric point comprised within a pH unit interval of less than 1;preferably less than 0.5; or preferably less than 0.4.

In another particular embodiment, the factor VII of the invention is arecombinant or transgenic factor VII. Preferably, the factor VII of thecomposition is produced by transgenic rabbits. In an embodiment, thefactor VII of the composition is an activated factor VII.

Another object of the invention relates to the use of the factor VIIcomposition for the treatment of bleeding episodes and for preventinghemorrhages occurring during surgical operations or invasive proceduresin the groups of the following patients:

-   -   in patients affected with congenital hemophilia with inhibitors        directed against the coagulation factors VIII or IX (hemophilia        A or B),    -   in patients affected with congenital hemophilia in which a        strong anamnestic response to the administration of factor VIII        or of factor IX is predictable.    -   in patients affected with acquired hemophilia,    -   and/or in patients having a congenital FVII deficiency.

Another object of the invention concerns a method for obtaining a factorVII composition according to the invention comprising the steps of:

-   -   (a) inserting a DNA sequence comprising a gene encoding for        factor VII in an embryonic non-human mammal, said gene being        under the transcriptional control of the beta-casein promoter,    -   (b) transferring the embryos obtained in step a) into the        oviduct of a female non-human mammal so that it develops into an        adult mammal,    -   (c) inducing lactation in the adult non-human mammal obtained in        step b) of the female type or in a female descendant of the        non-human mammal wherein the gene and the promoter are present        in its genome,    -   (d) collecting milk of said non-human mammal, and    -   (e) purifying the factor VII present in the milk collected.

Another object of the invention relates to a method for formulating afactor VII composition comprising the mixing of the factor VIIcomposition according to the invention with a buffer solution, pHadjustment if required, filtration and then drying if necessary in orderto obtain the solid form.

Other features and advantages of the present invention will becomeapparent upon reading the detailed description which follows and of thepreferred embodiment of the invention, given as an example and withreference to appended figures.

CAPTION OF THE FIGURES

FIG. 1: Method for purifying and extracting the factor VII of theinvention.

FIG. 2: Mass spectrum of the N-terminal peptides [Ala₁-Arg₃₆] and[Ala₁-Lys₃₂] of the factor VII of the invention.

FIG. 3: Charge profiling of the N-glycan forms of the factor VII of theinvention obtained by ultra-high performance liquid chromatographyhaving an anion exchanger resin coupled with detection by fluorescence(«AEX-UPLC/FD»)

FIG. 4: Comparison of the isoelectric points of the factor VII of theinvention and of a recombinant factor VII (Novoseven®) obtained by theisoelectric focusing method («IEF»).

FIG. 5: Profiling of charge of N-glycan forms of Factor VII of theinvention, of Factor VII from WO2007/138199 and a recombinant Factor VII(Novoseven®) obtained by ultra high performance liquid chromatographyhaving an anion exchange resin coupled to detection by fluorescence(“AEX-UPLC/FD”)

FIG. 6: Expression vector used for the generation of non-human mammalsexpressing human recombinant FVII according to the invention.

DETAILED DESCRIPTION

The factor VII composition chemically and physically stable at roomtemperature and easy to formulate according to the present invention ischaracterized in that the factor VII molecules which make it up, have asubstantially homogeneous isoelectric point.

Advantageously, the factor VII composition according to the inventioncomprises factor VII molecules, in which the factor VII molecules have asubstantially homogeneous isoelectric point.

By «factor VII» or «FVII», is meant the polypeptides comprising thesequence 1-406 of the human factor VII of the wild type (as for exampledescribed in U.S. Pat. No. 4,784,950 A of ZymoGenetics Inc.), or of thefactor VII derived from another species (for example, bovine, porcine,rabbit, caprine, murine species). It further comprises natural allelicvariations of the factor VII which may exist. The term «factor VII» alsoincludes the variants of FVII which have the same activity or a higherbiological activity as compared with the activity of the wild form,these variants notably including the polypeptides which differ from wildFVII by insertion, deletion or substitution of one or several aminoacids.

The term of «factor VII» or «FVII» also comprises the non-cleaved factorVII (zymogen) and the activated factor VII («factor VIIa» or «FVIIa»).In a preferred embodiment of the invention, the factor VII used in thecomposition according to the invention is preferably activated.

By the expression «biological activity of the factor VIIa», isunderstood the capability of FVIIa of generating thrombin, for exampleat the surface of activated platelets. The activity of the factor VII inthe composition according to the invention may be evaluated in differentways.

The biological activity of FVIIa may for example be quantified bymeasuring the capability of an FVII composition of promoting bloodcoagulation by using a FVII- and thromboplastin-deficient plasma, asdescribed for example in U.S. Pat. No. 5,997,864. In this test, thebiological activity is evaluated relatively to a control sample and isconverted into «FVII units» by comparison with a pooled standard humanserum containing 1 unit/ml of Factor VII activity. Alternatively, thebiological activity of the factor VII may be quantified (i) by measuringthe capability of the Factor Vila of producing the factor Xa in a systemcomprising TF encompassed in a lipid membrane and some Factor X (Perssonet al. J. Biol. Chem. 272:19919-19924, 1997): (ii) by measuring thehydrolysis of the factor X in an aqueous system (see «General methods»below); (iii) by measuring the physical bond of FVIIa to TF via surfaceplasmon resonance (Persson, FEBS letts, 413:359-363, 1997), (iv) bymeasuring the hydrolysis of a synthetic substrate or (v) by measuringthe generation of thrombin in an in vitro system independent of TF.

In a particular embodiment, the factor VII of the invention is arecombinant factor VII. By «recombinant factor VII», is meant any factorVII stemming from genetic engineering and resulting from the expressionof the corresponding gene in any microorganism, plant, transgenic plant,animal or transgenic animal. By microorganism, is meant any bacterial,fungal, viral or cellular system. The recombinant factor VII may also beproduced from eukaryotic cells in culture such as plant or mammal cells,for example animal or human cells.

Thus, the FVII of the invention is derived from transcription andtranslation of a DNA molecule encoding FVII in a host cell. Therecombinant FVII of the invention may be obtained using standardtechniques, well known to those skilled in the art, allowing expressionof a protein in a biological system.

More specifically, the term “recombinant FVII” means any FVII obtainedby genetic recombination and expressed in a cell line that is cultured.Examples are the following lines: BHK (Baby, Hamster Kidney) and notablyBHK tk “ts13 (CRL 10314, Waechter and Baserga, Proc Natl Acad Sci USA79:1106-1110, 1982 . . . ), CHO (ATCC CCL 61), COS-I (ATCC CRL 1650),HEK293 (ATCC CRL 1573. Graham et al, J. Gen. Virol 36:59-72, Gen 1977),Rat Hep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma, ATCCCRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469(ATCC CCL 9.1) and DUKX cells (CHO cell line) (Urlaub and Chasin, ProcNatl Acad Sci USA 77:4216-4220 1980), 3T3 cells, Namalwa cells, or BHKcells adapted to culture without serum (U.S. Pat. No. 6,903,069).

In a particular embodiment, the cell line is modified so as to producecompositions according to the invention, for example by modulating theexpression of a glycosyltransferase, in particular over-expressing asialyltransferase.

In a particular embodiment, the factor VII of the invention is atransgenic factor VII. By «transgenic factor VII», is meant anyrecombinant factor VII obtained from a transgenic animal.

By «transgenic animal», is meant any non-human animal having amodification of its genome intended for allowing expression of thefactor VII according to the invention. The modification of the genomeadvantageously results from an alteration, a modification or theinsertion of a gene. This modification may be due to the action ofconventional altering agents or mutagens or else carried out by directedmutagenesis. The modification of the genome may also result frominsertion of gene(s) or replacement of gene(s) in its (their) wild ormutated form. In a preferred embodiment, the factor VII of the presentinvention is obtained from milk of a transgenic animal. A transgenicanimal according to the invention be selected from rabbits, goats, cows,camels, hamsters, mice, rats, horses, sows, dromedaries, ewes, llamas,the list not being a limitation. Preferably, the factor VII of thepresent invention is obtained from the milk of transgenic rabbits. Thesecretion of the factor VII by the mammary glands, allowing itssecretion in the milk of the transgenic mammal, involves the control ofthe expression of the factor VII in a tissue-dependent way. Such controlmethods are well known to one skilled in the art. The control of theexpression is carried out by sequences allowing expression of theprotein towards a particular tissue. These are notably WAP, beta-casein,beta-lactoglobulin promoter sequences and signal peptide sequences, thelist not being a limitation. Particularly advantageously, expression inthe mammary glands of the rabbits is performed under the control of thepromoter of beta-casein, well known to those skilled in the art.

A method for producing a recombinant protein in the milk of a transgenicanimal can include the following steps: a synthetic DNA moleculecomprising a gene coding for human FVII, this gene being under thecontrol of a promoter of a protein naturally secreted into the milk isintegrated into an embryo of a non-human mammal. The embryo is thenplaced in a female mammal of the same species. Once the mammal derivedfrom the embryo has sufficiently developed, the lactating mammal isinduced, then the milk is collected. The milk then contains the FVII ofinterest secreted by the transgenic animal.

A protein preparation example in the milk of a mammal female other thanthe human being is given in the application EP 0 527 063 B 1 (InstitutNational de la Recherche Agronomique (National Institute for AgronomicResearch)), the teaching of which may be used again for producing thefactor VII of the invention.

In a preferred embodiment, the factor VII according to the invention isproduced in the milk of transgenic rabbits. In particular, a plasmidcontaining the beta-casein promoter is prepared by introduction of asequence comprising the promoter of the beta-casein gene, the plasmidbeing constructed so as to receive a foreign gene placed under thecontrol of this promoter. The gene encoding for the human factor VII isintegrated, and placed under the dependency of the beta casein promoter.The plasmid containing the promoter and the sequence encoding theprotein of interest is digested with restriction enzymes to release theDNA fragment containing the beta-casein promoter and the sequence ofhuman FVII. After purification, fragments are introduced bymicroinjection into the male pronucleus of wild-type (WT) rabbitembryos. Embryos are then cultivated before being transfer into theoviduct of wild-type females that are hormonally prepared. At droppingof the females, the descendants are evaluated by PCR to determinetransgenic animals. The copy number of the transgenes and theirintegrity is revealed by Southern techniques from DNA extracted from thetransgenic baby rabbits obtained. Concentrations in human milk oftransgenic animals FVII are measured using specific radioimmunoassaytests.

In a particular embodiment, the composition of factor VII according tothe invention is obtained by the method comprising the steps of:

(a) inserting a DNA sequence comprising a gene encoding the factor VIIin an embryonic non-human mammal, said gene being under thetranscriptional control of the beta-casein promoter,

(b) transferring the embryos obtained in step a) into the oviduct of afemale non-human mammal so that it develops into an adult mammal,

(c) inducing lactation in the adult non-human mammal obtained in step b)of the female type or in a female descendant of the non-human mammalwherein the gene and promoter are present in its genome,

(d) collecting the milk of said non-human mammal, and

(e) purification of factor VII present in the milk collected.

In a particular embodiment, the adult non-human mammal or the femaledescendant of the non-human mammal is selected notably for its capacityto produce factor VII compositions according to the invention. Thetransgenic animal can also be selected according to others criteria, asfor example the presence of only one integration site and/or theintegrity of the product.

In a particular embodiment, the insertion of a DNA sequence comprising agene coding for factor VII in an embryonic non-human mammal, said genebeing under the transcriptional control of the beta-casein promoter isby microinjection more particularly in the male pronucleus of a rabbitembryo.

By “embryo”, is meant the male or female pronucleus of an embryo ofnon-human mammal, more particularly of rabbit, fertilized.

By «isoelectric point» or «pI», is meant the pH for which the netelementary charge of the factor VII or factor VIIa molecule is zero,i.e. the pH at which the molecule is electrically neutral (zwitterionicform). The isoelectric point of the factor VII according to theinvention may be measured by applying a technique well known to oneskilled in the art, such as isoelectric focusing («IEF»). Thiselectrophoretic technique allows separation of the proteins on the basisof their isoelectric point. It consists in a migration induced by auniform electric current, of proteins in a pH gradient until they reacha pH equivalent to their specific isoelectric point, a moment at whichthey cease migration since their net charge is zero. IEF gels are usedfor determining the isoelectric point of a given protein and fordetecting minor changes of the latter due to post-translationalmodifications such as γ-carboxylations, phosphorylations orglycosylations.

By «substantially homogeneous», is meant that at least 90%, preferablyat least 95% of the factor VII molecules of the composition have anisoelectric point comprised in a pH unit interval of less than or equalto 1.2. In another embodiment of the invention at least 50%, preferablyat least 55%, preferably 60%6 of the factor VII molecules of thecomposition have an isoelectric point comprised in a pH unit interval ofless than 1; preferably less than 0.5. In another preferred embodiment,at least 50%, preferably at least 55%, preferably 60% of the factor VIImolecules of the composition have an isoelectric point comprised in a pHunit interval of 0.4.

By «N-glycan forms», is meant the whole of the N-glycan forms present onboth N-glycosylation sites of the factor VII of the invention.

N-glycan forms are said to be monocharged, if their total charge isequal to 1. In the sense of the present invention, by «charge», is meanta phosphate group, a sulfate group, or a sialic acid molecule. Thus, theN-glycan forms are said to be monocharged, if they only contain aphosphate group or a sulfate group or a sialic acid molecule. As opposedto the term of «monocharged», the term of «bicharged» means that thetotal charge borne by the N-glycan forms is equal to 2, i.e. they havetwo charges selected from a phosphate group, a sulfate group and/or asialic acid molecule. In other words, the N-glycan bicharged forms haveeither a sialic acid molecule and a phosphate group, or a sialic acidmolecule and a sulfate group, or two sialic acid molecules, or twophosphate groups, or two sulfate groups, or a phosphate group and asulfate group. The term of «neutral» as for it, means that the N-glycanforms do not contain any charge.

The charge of the N-glycan forms of the factor VII according to theinvention may be measured by applying a technique well known to oneskilled in the art, notably by ultra-high performance liquidchromatography with an anion exchanger resin coupled with detection byfluorescence (AEX-UPLC/FD). With this method it is possible to separatethe different N-glycan forms according to their apparent charge (see inparticular Hermentin et al., Glycobiology, Vol. 6, No. 2, 1996). Withinthe scope of anion exchange chromatography, a positively charged resinis used as a stationary phase. These positively charged resins generallyconsist of a cross-linked polymer or gel, on which are graftedpositively charged groups. In an advantageous embodiment of theinvention, a low anion exchange column of the amino propyl type is used.

The applicant has more particularly demonstrated that the substantiallyhomogeneous isoelectric point of the composition of factor VII accordingto the invention results from the combination of the glycosylation andγ-carboxylation properties of the FVII molecules which make it up.

The factor VII molecules which form the composition according to theinvention include, similarly to the human plasma factor VII, twoN-glycosylation sites in the positions 145 and 332, and 2 sites ofO-glycosylation, in the positions 52 and 60. Each of these sites mayreceive «glycans» or «sugar chains» or «oligossacharide chains». TheN-glycosylated and/or O-glycosylated oligosaccharide chains borne byeach Factor VII molecule of the composition may differ from one moleculeto another. However, it is possible to measure the distribution of eachunit in the factor VII composition according to the invention by usingtechniques known to one skilled in the art.

In the case of the factor VII composition according to the invention, itappears that among all the N-glycan forms of the factor VII of thecomposition, at least 60% of the N-glycan forms, preferably at least65%, preferably at least 70%, preferably at least 75%, preferably atleast 80%, preferably at least 85%, preferably at least 90%, preferablyat least 95%, are monocharged. In a preferred embodiment, the factor VIImolecules having monocharged N-glycan forms represent between 60% and95% of the factor VII molecules of the composition, preferably between65% and 90% of the factor VII molecules of the composition, preferablybetween 70% and 85% of the factor VII molecules of the composition,preferably between 70% and 80% of the factor VII molecules of thecomposition, preferably between 74% and 77% of the factor VII moleculesof the composition.

In the case of the composition of Factor VII according to the invention,it appears that among all the N-glycan forms of Factor VII of thecomposition, between 3% and 10%, preferably between 3% and 7% of theN-glycan forms are neutral.

In the case of the composition of Factor VII according to the invention,it appears that among all the N-glycan forms of Factor VII of thecomposition, between 15% and 25%, preferably between 18% and 22% of theN-glycan forms are bicharged.

In the case of the composition of factor VII according to the invention,it appears that among all the N-glycan forms of factor VII of thecomposition, less than 5%, preferably less than 2%, preferably less than1% of the N-glycan forms are tricharged.

In the case of the composition of factor VII according to the invention,it appears that among all the N-glycan forms of factor VII of thecomposition, less than 5%, preferably less than 2%, preferably less than1% of the N-glycan forms are tetracharged.

For the purposes of the invention, it is understood that the percentagescited above do not therefore take into account glycan forms involved inO-glycosylation.

Moreover, the factor VII molecules forming the composition of theinvention only comprise sialic acids bound through bounds of the α2-6type. The applicant also noticed that the fucose content of the factorVII had not effect on the isoelectric point of the composition of factorVII.

In the case of the factor VII composition according to the invention, itappears that at least 60%, preferably at least 65%, preferably at least70% of the N-glycan forms of the factor VII of the composition aremonosialylated complexes.

The complex N-glycan forms are forms well known to one skilled in theart (see in particular. Kornfeld R et al, Annual Review of Biochemistry.1985; 54:631-64. Assembly of asparagine-linked oligosaccharides).

In a particular embodiment, at least 10%, preferably at least 15%,preferably at least 20%, preferably at least 25% of the N-glycan formsof the factor VII of the composition are high Mannose/hybrid.

By «γ-carboxylation», is meant the product of a biochemical reactionaiming at transforming a plurality of glutamic acid residues present inthe peptide sequence of the factor VII into carboxy-glutamic acid or“GLA” residues.

Advantageously, the factor VII molecules which form the compositionaccording to the invention have, for at least 80% of them,γ-carboxylation on nine residues of glutamic acid. In anotherembodiment, at least 85% of said molecules have γ-carboxylation on nineresidues of glutamic acid. In another embodiment, between 85% and 100%,preferably between 90% and 100%, preferably between 95% and 100% of saidmolecules have γ-carboxylation on nine residues of glutamic acid.Advantageously, the γ-carboxylation level on the residue of glutamicacid 35 (Glu₃₅) of the factor VII molecules of the composition is lessthan 20%. In another embodiment, the γ-carboxylation level of theresidue Glu₃₅ is less than 15%, preferably less than 10%, preferablyless than 5%.

The applicant surprisingly discovered that it is the specificcombination of the level of the monocharged N-glycan forms and of theγ-carboxylation level, which is responsible for the substantialhomogeneity of the isoelectric points of the factor VII moleculesforming the composition according to the invention.

The FVII composition according to the invention may advantageously beformulated without causing precipitation of the FVII molecules whichmake it up. Indeed, it is known that at the isoelectric point of themolecule, the latter aggregates and precipitates. The factor VIImolecules which form the composition according to the invention have anisoelectric point comprised between 6.6 and 7.0. The result of this isbetter stability of the factor VII composition according to theinvention, notably when the latter is formulated at a pH below theisoelectric point, and in particular at a pH of 6. The improvement ofthe stability of the factor VII composition according to the inventiongives the possibility of avoiding the electrostatic interactionsresponsible for precipitation and aggregation phenomena of the solubleand insoluble type, as well of avoiding the loss of active raw materialsand therefore a lowering of the yield resulting in a quantitative lossof active ingredients and therefore potentially in a loss of activity.

Another object of the invention concerns a purification method for theactivated factor VII according to the invention.

The milk of transgenic rabbits is obtained from the line of transgenicrabbits R69. The frozen milk of transgenic rabbits is defrosted andconcentrated in the form of a milk pool from transgenic rabbits.

The thereby obtained milk pool from transgenic rabbits is then subjectto a clarification step using a filter in depth having a porosity of 0.2μm, in order to remove the lipids and insoluble compounds. The therebyclarified milk is then subject to a viral inactivation step by treatmentwith a solvent of the detergent type, for example Polysorbate 80 orTri-n-Butyl Phosphate at 25° C.±2° C. for at least two hours. Such atreatment notably gives the possibility of efficiently inactivatingviruses, and in particular the viruses of the non-enveloped virusestype. The clarified and virally inactivated milk is then subject to anaffinity chromatography step using a specific affinity ligand of thefactor VII/factor VIIa. The eluate of factor VII obtained at the end ofthis chromatography step is then subject to an ultra-filtration andformulation step, thereby giving the possibility of obtaining anintermediate factor VII concentrate having a purity of 95%.

The intermediate factor VII concentrate is then subject to a filtrationstep using a filter having a porosity from 0.2 μm to 0.2 μm followed bya nanofiltration step on filters having a porosity of 20 nm and then of15 nm. The thereby obtained product and containing the factor VII isthen subject to a chromatography step of the Q Sepharose XL gel type andthen to a chromatography step of the CHT-I type followed bychromatography of the SEC Superdex 200 type. The thereby obtainedconcentrate of factor VII is then subject to a stabilization step andthen to a filtration step on a filter having a porosity of 0.2 μm.

The thus described method gives the possibility of obtaining a factorVII concentrate having a purity of about 99.9995%.

In a preferred embodiment, the method of purification and extraction ofthe factor VII is the one described in application EP12305882.

Another object of the present invention relates to a method forformulating an activated factor VII composition according to theinvention.

Advantageously, the formulation method used is the one described inapplication WO2010/149907.

This method notably comprises the mixing of the activated factor VIIcomposition according to the invention with a buffer solution,adjustment of the pH if required, filtration and drying if necessary forobtaining the solid form.

In a preferred embodiment, the step for mixing the activated factor VIIcomposition with a buffer solution is applied in chromatography of thegel permeation type. The term of «buffer solution» includes at least onehydrophilic amino acid or bearing a positively charged side chain, andan alkaline metal salt, an earth alkaline metal salt, or a transitionmetal salt. Mention may notably be made of trisodium citrate, calciumchloride, or zinc chloride. Preferably the salt used is preferentiallysodium citrate or calcium chloride. Advantageously, the buffer solutionalso contains at least one hydrophobic amino acid.

Advantageously, the buffer solution comprises one or several of theconstituents selected from:

-   -   one salt, preferably a citrate salt, preferably trisodium        citrate;    -   an amino acid, or a hydrophilic amino acid salt, preferably a        hydrophilic amino acid salt, preferably arginine hydrochloride        and/or lysine hydrochloride;    -   an amino acid or a hydrophobic amino acid salt, preferably a        hydrophobic amino acid, preferably isoleucine and/or glycine.

Finally, the composition of the invention may comprise one or severaldetergents of the non-ionic type such as polysorbates, polyoxamers,polyoxyethylene alkyl ethers, an ethyl/polypropylene block copolymer andpolyethylene glycol. Advantageously, the preferred detergents arepolysorbate 80 and polysorbate 20.

Advantageously, the factor VII composition obtained at the end of themixing step with the buffer solution comprises:

-   -   the factor VII according to the invention, preferably in        activated form;    -   from 10 to 40 g/l of arginine, optionally as a hydrochloride;    -   from 4.2 to 6.6 g/l of isoleucine;    -   from 0.6 to 1.8 of lysine;    -   from 0.6 to 1.8 g/l of glycine;    -   from 1 to 2 g/l of trisodium citrate dehydrate or from 0 to 0.2        g/l of calcium chloride dihydrate; and if necessary from 0 to        0.5 g/l of polysorbate 80.

According to a particular example, the factor composition obtained atthe end of the mixing step with the buffer solution comprises somefactor VII (preferably as factor VIIa) at a concentration from 0.2 to 2g/l, arginine hydrochloride at 24 g/l, isoleucine at 6 g/l, trisodiumcitrate dehydrate at 1.5 g/l, glycine at 1.2 g/l, lysine hydrochlorideat 1.2 g/l and/or polysorbate 80 at 0.07 g/l.

In a particular example, the composition of factor obtained after thestep of mixing with the buffer solution comprises factor VII (preferablyin the form of factor VIIa) at 0.2 to 2 g/l, arginine hydrochloride at24 g/l, isoleucine at 6 g/l, dehydrated trisodium citrate at 1.5 g/l,glycine at 1.2 g/l, lysine hydrochloride at 1.2 g/l, and/or polysorbate80 at 0.09 g/l.

According to another particular example, the factor VII compositionobtained at the end of the mixing step with the buffer solutioncomprises the factor VII (preferably as factor VIIa) at a concentrationfrom 0.2 to 2 g/l, arginine hydrochloride at 34 g/l, calcium chloridedihydrate at 0.15 g/l, isoleucine at 6 g/l.

Preferably, the factor VII composition according to the invention iswithout any sugar, polyol or methionine. The sugars to be avoidednotably include, in addition to saccharose, di- and tri-saccharides andpolysaccharides, such as dextrose, lactose, maltose, trehalose,cyclodextrins, maltodextrins and dextrans. The polyols to be avoidednotably include sorbitol and xylitol in addition to mannitol.

Still preferably, the composition is without glycylglycine. Stillpreferably, the composition is without glycylglycine.

According to a preferred embodiment, the composition of the invention isfurther without any antioxidant. The antioxidants for example includeone or several of the following compounds: homocysteine, cysteine,cystathionine, methionine, glutathion.

The pH of the solution before drying is preferably comprised between 4.0and 9.0, more particularly in the ranges 4.0 and 8.0; 4.0 and 7.5; 4.5and 7.5; 5.0 and 7.5; 5.5 and 7.0; 6.0 and 7.5; 6.5 and 7.5.

Drying is a method for removing water extensively. This is dehydrationaiming at removing as much water as possible. This phenomenon may benatural or forced. This drying may be achieved by freeze-drying,atomization and cryoatomization techniques. The preferred method forobtaining the solid form of the composition for pharmaceutical useaccording to the invention is freeze drying. Freeze-drying methods arewell known to one skilled in the art, see for example [Wang et al.,Lyophilization and development of solid protein pharmaceuticals,International Journal of Pharmaceutics, Vol. 203, p 1-60, 2000].

Other suitable methods for reducing the humidity level of the watercontent of the composition may be contemplated. Preferably, the humiditylevel is less than or equal to 3% by weight, preferably less than orequal to 2.5% by weight, preferably less than or equal to 2%, preferablyless than or equal to 1.5%.

Preferably, the formulation method further comprises a step foreliminating or inactivating infectious agents, for example by dryheating the lyophilizate.

Preferably, the formulation method further comprises a step forfiltering the factor VII composition and/or a step for freeze-drying theformulated factor VII composition.

The factor VII composition according to the invention is particularlyadvantageous when it is formulated according to the method describedabove. Indeed, the formulation method according to the invention appliedto the factor VII composition according to the invention allowsimprovement of its stability.

Another object of the invention relates to the use of the factor VIIcomposition of the invention as a drug, for the treatment of bleedingepisodes.

Another object of the invention relates to the use of the factor VIIcomposition according to the invention for preventing hemorrhagesoccurring during surgical operations and invasive procedures.

Another object of the invention is a pharmaceutical compositioncomprising the factor VII composition according to the invention and anexcipient and/or a pharmaceutically acceptable carrier.

The excipient may be any solution, such as a saline, physiological,isotonic or buffered solution as well as any suspension, gel or powdercompatible with pharmaceutical use and known to one skilled in the art.The compositions according to the invention may further contain one orseveral agents or carriers selected from dispersants, solubilizers,stabilizers, surfactants and preservatives. On the other hand thecomposition according to the invention may comprise other active agentsor ingredients.

Moreover, the compositions may be administered in different ways and indifferent forms. The administration may be carried out via anyconventional route for this type of therapeutic approach, such asnotably via a systemic route, in particular by intravenous, intradermal,intra-tumoral, sub-cutaneous, intra-peritoneal, intramuscular orintra-arterial injection. Mention may be made for example ofintra-tumoral injection or injection in an area close to the tumor orirrigating the tumor.

The dosages may vary according to the number of administrations, to thecombination with other active ingredients, to the development stage ofthe pathology, etc.

Of course, the present invention is not limited to the described andillustrated examples and embodiments, but it is subject to manyalternatives accessible to one skilled in the art.

EXAMPLES Example 1: Production of Transgenic Rabbits Producing HumanFVII Protein in their Milk

An expression vector comprising a beta-globin insulating sequence fromchicken, the control region of goat beta-casein at 5′, an optimised cDNAsequence for the expression in mammal cells coding for human FVII andthe non-translated region of beta-casein at 3′ was prepared in aSuperCos backbone. The assembly in FIG. 6 represents the constructionvector.

The transgene was prepared from the digestion of the construction vectorwith the NotI restriction endonuclease to release the SuperCos backboneand linearized the expression vector. The resulting fragments wereseparated by gel electrophoresis, purified from the gel and used formicroinjection.

To produce transgenic rabbits, the microinjection of the transgene intomale pronuclei of embryos fertilized collected from WT rabbits wasperformed. After a period of in vitro culture of a few hours, themicroinjected male pronuclei of embryos were transferred into theoviduct of female non-transgenic rabbits hormonally prepared. Aftergestation, the progeny are obtained. A genetic analysis allows theevaluation of the presence of the human factor VII transgene by PCTanalysis using primers specific from human factor VII sequence. Thepresence of the transgene and its integrity has been revealed by theSouthern blotting technique from DNA extracted from the transgenic babyrabbits obtained. A phenotypic analysis allows the evaluation of thehuman factor VII concentration expressed in the milk of transgenicfemale rabbits. This measure is realized by enzyme immunoassay (ELISA)using commercial reagents (Diagnostica Stago). Briefly, the factor VIIto be assayed is captured by a human anti-factor VII antibodyimmobilized on a solid phase. The factor VII fixed was then recognizedby a known immuno-peroxidase conjugate. The rate of bound peroxidase ismeasured by its activity on the ortho-phenylenediamine substrate in thepresence of hydrogen peroxide. The intensity of the coloration, afterstopping the reaction with a strong acid, is a function of the amount offactor VII initially present in the sample.

Example 2: Purification and Extraction of the Obtained Factor VII

The method for purifying and extracting the factor VII applied in thisexample is described below.

The milk of transgenic rabbits is obtained from the line of transgenicrabbits R69. The frozen milk of transgenic rabbits is defrosted andconcentrated in the form of a milk pool from transgenic rabbits.

The thereby obtained milk pool from transgenic rabbits is then subjectto a clarification step using a filter in depth having a porosity of 0.2μm, in order to remove the lipids and insoluble compounds. The therebyclarified milk is then subject to a viral inactivation step by treatmentwith a solvent of the detergent type, for example Polysorbate 80 orTri-n-Butyl Phosphate at 25° C.±2° C. for at least two hours. Such atreatment notably gives the possibility of efficiently inactivatingviruses, and in particular the viruses of the non-enveloped virusestype. The clarified and virally inactivated milk is then subject to anaffinity chromatography step using a specific affinity ligand of thefactor VII/factor VIIa. The eluate of factor VII obtained at the end ofthis chromatography step is then subject to an ultra-filtration andformulation step, thereby giving the possibility of obtaining anintermediate factor VII concentrate having a purity of 95%.

The intermediate factor VII concentrate is then subject to a filtrationstep using a filter having a porosity from 0.2 μm to 0.2 μm followed bya nanofiltration step on filters having a porosity of 20 nm and then of15 nm. The thereby obtained product and containing the factor VII isthen subject to a chromatography step of the Q Sepharose XL gel type andthen to a chromatography step of the CHT-I type followed bychromatography of the SEC Superdex 200 type. The thereby obtainedconcentrate of factor VII is then subject to a stabilization step andthen to a filtration step on a filter having a porosity of 0.2 μm.

The thereby described method gives the possibility of obtaining a factorVII concentrate having a purity of about 99.9995%.

The method for extracting and purifying factor VII is described ingreater detail in European application EP12305882.

In the examples appearing in the present application, the recombinantactivated factor VII available on the market (Novoseven®), produced bygenetic engineering from BKH kidney cells of newborn hamsters is called«FVII-rec».

Example 3: Study of the Chare Profiling of N-glycans

A Identification and Quantification of N-Bound Oligosaccharides by HighPerformance Liquid Chromatography with Normal Phase Polarity in theHILIC Mode Coupled with Fluorimetric Detection (HILIC-HPLC/FD), aso-Called «HILIC» Method.

The hydrophilic interaction chromatography method (HILIC or HydrophilicInteraction Chromatography) is an alternative of normal phase (NP)chromatography and allows sufficient separation of the polar species byavoiding the drawbacks related to the use of solvents which are notmiscible in water. The stationary phase is a polar material generallybased on silica or polymeric supports grafted through a functional groupsuch as a cyano, amino, diol, amide group etc. and the mobile phase isorganic and contains water as a strong eluent.

The factor VII is first of all subject to enzymatic digestion withPNGase F in order to specifically release the N-glycan derivatives.

The latter are isolated by extraction on a solid phase (SPE) and thenlabeled with a fluorophore: 2-aminobenzamide (2-AB) according to Biggeet al. (Bigge, J. C., et al., Non-selective and efficient fluorescentlabeling of glycans using 2-aminobenzamide and anthranilic acid, Anal.Biochem., 230, 229-238 (1995)). Analysis of the N-glycan derivatives isfinally carried out with HILIC-HPLC/FD according to Guile et al. (Guile,G. R., et al., A rapid and high-resolution high-performance liquidchromatographic method for separating glycans mixtures and analyzingoligosaccharide profiles, Anal. Biochem., 240, 210-226 (1996)).Detection is ensured by fluorescence at the wavelengths of aexcitation=330 nm and λ emission=420 nm.

The results obtained for the factor VII of the invention as well as forthe FVII-rec are shown in the tables below.

TABLE 1 Quantification of neutral, nanocharged and bicharged N-glycanforms of the factor VII of the invention obtained by the HILIC methodN-glycan forms FVII FVII FVII (expressed in %) (Batch 1) (Batch 2)(Batch 3) Neutral 3.6 4.3 3.9 Monocharged 75.5 74.6 75.3 Bicharged 21.021.2 20.6

The results obtained by the HILIC method show that the factor VII of theinvention has a level of neutral N-glycan forms comprised between 3.6%and 4.3%, of monocharged N-glycan forms comprised between 74.6% and75.5% and of bicharged N-glycan forms comprised between 20.6% and 21.2%.

TABLE 2 Quantification of the neutral, monocharged and discharged,tricharged, tetracharged N-glycan forms of the factor VII-rec obtainedby the HILIC method. FVII-rec N-glycan forms (results from thepublication (expressed in %) FVII-rec of Klausen et al.*) Neutral 11.39.4 Monocharged 25.1 27.9 Bicharged 55.1 51.2 Tricharged 7.3 11.6Tetracharged 0.7 0 (*Klausen et al., Analysis of the site-specificasparagine-linked glycosylation of recombinant human coagulation factorVIIa by glycosidase digestions, liquid chromatography, and massspectrometry, Mol Biotechnol. 1998 June; 9(3): 195-204.).

The results obtained by the HILIC method show that the neutral N-glycanforms of FVII-rec represent 11.3% of the N-glycan forms of the FVII-rec,that the monocharged N-glycan forms represent 25.1% of the N-glycanforms of the FVII-rec, and that the bicharged N-glycan forms represent55.1% of the N-glycan forms of the FVII-rec. Further, the factorFVII-rec has a non-negligible amount of tricharged and tetrachargedforms.

The results thereby obtained by the HILIC method are therefore compliantwith those shown in the publication of Klausen et al.

B Identification and Quantification of N-Bound Oligosaccharides by theAEX/UPLC/FD Method

The separation of the N-glycans according to their charge is carried outby AEX-UPLC/FD profiling of the derived N-glycans labeled with 2-AB. Theoperating procedure for preparing the derived N-glycans labeled with2-AB is identical with the one described earlier for analyzing N-glycansby HILIC-HPLC/FD.

The profiling of the charge of the N-glycans of FVII by AEX-UPLC/FD wasachieved by means of a low anion exchange column of the aminopropyltype. The glycans are separated according to their charge by applying anincreasing gradient of ammonium formate. The detection of the derivativeis carried out at the following wavelengths: excitation wavelength: 330nm and emission wavelength: 420 nm. The profile obtained by AEX-UPLC/FDfor the FVII (batch 1) is shown in FIG. 3.

The obtained results are shown in Table 3 below.

TABLE 3 Quantification of the neutral, monocharged and bichargedN-glycan forms obtained for the factor VII of the invention by theAEX-UPLC/FD method FVII FVII FVII (batch 1) (batch 2) (batch 3)Retention expressed expressed expressed N-glycan forms time in % in % in% Neutral 2-6 mm 4.0 4.2 3.4 Monocharged 11-14 min 76.5 76.9 77.2Bicharged 17-20 min 19.6 18.9 19.4

It is observed that the percentage of monocharged N-glycan forms of thethree batches of FVII according to the invention is comprised between 76and 78%.

Both of these orthogonal methods (HILIC and AEX) show that at least74.6% of the N-glycan forms of factor VII of the invention aremonocharged, independently of the measuring method used.

C Identification, Quantification and Comparison of N-LinkedOligosaccharides by the AEX/UPLC/FD Method for Factor VII of theInvention, Factor VII from Application WO2007/138199 and Factor VII-Rec.

The procedure used is the same as that described in Example 3. Profilingof N-glycan charge of FVII by AEX-UPLC/FD was performed on the factorVII from the invention, factor VII derived from applicationWO2007/138199 and factor VII-rec.

Glycan profiles are presented in FIG. 5. FIG. 5A shows the glycanprofile of FVII resulting from application WO2007/138199. FIG. 5B showsthe glycan profile of FVII of the invention and FIG. 5C shows the glycanprofile of FVII-rec.

TABLE 4 Quantification of neutral, monocharged and bicharged N-glycanicforms obtained by the AEX-UPLC/FD method FVII from FVII (origi- theinven- nating from tion application Factor (batch 4) WO2007/138199)VII-rec N-glycanic Retention expressed expressed expressed forms time in% in % in % Neutral (Z₀) 3-5 min 7.0 3.0 7.0 Monocharged 5-7 min 73.051.0 29.0 (Z₁) Bicharged (Z₂) 7-10 min 20 39.0 54.0 Tricharged (Z₃)10-12 min 0.0 7.0 9.0 Tetracharged 12-14 min 0.0 0.0 1.0 (Z₄)

As regards the FVII resulting from application WO2007/138199, theresults obtained using the AEX/UPLC/FD method show:

-   -   that neutral N-glycan forms of FVII resulting from application        WO2007/138199 represent 3% of N-glycan forms of this FVII,    -   that monocharged N-glycan forms represent 51% of the N-glycan        forms of this FVII and,    -   that bicharged N-glycan forms represent 39% of the N-glycan        forms of this FVII,    -   a non-negligible amount of tricharged forms.

The results obtained using the AEX/UPLC/FD method show that neutralN-glycan forms of FVII-rec represent 7.0% of N-glycan forms of FVII-rec,that monocharged N-glycan forms represent 29.0% of the N-glycan forms ofFVII-rec, and that bicharged N-glycan forms represent 54.0% of theN-glycan forms of FVII-rec. In addition, the factor VII-rec has anon-negligible amount of tricharged and tetracharged forms.

Example 4: Identification, Quantification and Comparison of the N-Glycan

Forms of factor VII according to the invention and factor VII resultingfrom PCT Application WO2007/138199 by the HILIC method

A Quantification of Factor VII N-Glycan Forms According to the InventionCarried Out Using the HILIC Method.

The quantification of the N-glycan forms of the factor VII according tothe invention is achieved by means of the HILIC method as described inExample 3.

The thereby obtained results are shown in Table 5.

TABLE 5 Quantification of the N-glycans obtained for the factor VIIcomposition of the invention. N-glycan forms FVII FVII FVII (expressedin %) (batch 1) (batch 2) (batch 3) Charge A1G0 0.5 0.8 0.5 0 Man5 1.31.5 1.4 0 Man4-A1G1 0.2 0.4 0.4 0 A2G2 1.4 1.1 1.2 0 A1G1S1 1.1 1.2 1.21 A2G2F 0.5 0.4 0.4 0 A2G1S1 0.5 0.7 0.3 1 Man4-A1G1S1 2.8 2.9 4.2 1Man6PGlcNAc 9.8 9.1 9.9 1 A2G2S1 43.3 41.8 41.1 1 A2G2S1-SO3H 0.9 0.90.9 2 Man5-A1G1S1 0.2 0.4 0.2 1 A2G2FS1 8.8 9.3 9.9 1 Man7PGlcNAc 2.83.2 3 1 A2G2FS1* 2 2.3 1.9 1 Man7PGlcNAc* 1.7 1.6 1.6 1 A2G2F2S1 2.2 2.22 1 A2G2S2 9.1 9.7 8.8 2 Man7(PGlcNAc)2 3.4 2.8 3.2 2 A2G2FS2 2.6 3.12.8 2 Man6PGlcNAc- 5 4.7 4.9 2 A1G1S1 High-Mannose/ 1.5 1.9 1.8 0Neutral Hybrid High-Mannose/ 17.3 17.2 18.9 1 Monocharged HybridHigh-Mannose/ 8.4 7.5 8.1 2 Bicharged Hybrid Total High- 27.2 26.6 28.8— Mannose/Hybrid Monocharged 75.2 74.7 75.4 1 N-glycan formsMonosialylated 58.8 58.4 57.3 1 Complex Bisialylated Complex 11.7 12.811.6 2 *Isomeric forms

Quantitative analysis of the different glycan structures (Table 5) showthat the N-glycan forms of the factor VII of the composition are inmajority monosialylated complexes (around some 60%). The N-glycan formsof the high mannose/hybrid type are present at a minimum of 25%.

B Quantification of the Factor VII N-Glvcan Forms Resulting fromApplication WO2007/138199 Factor VII Carried Out Using the HILIC Method.

Quantification of factor VII N-glycan forms resulting from applicationWO2007/138199 is carried out using the HILIC method, as described inExample 3. The results thus obtained are shown in Table 6.

TABLE 6 Quantification of N-glycans obtained for the factor VIIcomposition resulting from application WO2007/138199 FVII originatingfrom N-glycan forms International application (expressed in %)WO2007/138199 Charge Man5 0.7 0 A2G2 0.3 0 A1G1S1 0.6 1 Man5-A1G1 0.3 0A2G2F 0.3 0 A2G1S1 + A1G1FS1 0.4 1 Man4-A1G1S1 0.8 1 Man6PGlcNAc 6.2 1Man6PGlcNAc* 1.4 1 A2G2S1 18.2 1 A2G2FS1 9 1 Man7PGlcNAc 1.6 1 A2G2FS1*4 1 Man7PGlcNAc* 0.6 1 Monocharged HM/Hyb 0.3 1 A2G2F2S1 9.7 1 A2G2S210.9 2 A3G3S1 0.7 1 Man7(PGlcNAc)2 2 2 A2G2FS2 15.9 2 A3G3F0-1S1 0.3 1A3G3F0-1S1* 0.9 1 A3G3F1-2S1 0.6 1 Man6PGlcNAc-A1S1 4.3 2 A3G3S2 1.8 2A3G3S2* 0.5 2 A3G3FS2 1 2 A3G3FS2* 1.1 2 A3G3F0-2S2 0.7 2 A3G3F0-2S2-30.7 3 A3G3S3 2.8 3 A3G3F0-2S3 1 3 A3G3F0-2S3* 0.3 3 High-Mannose/NeutralHybrid 1 0 High-Mannose/Monocharged Hybrid 10.9 1 High-Mannose/BichargedHybrid 6.3 2 Total High-Mannose/Hybrid 18.2 — Monocharged N-glycannicforms 55.3 1 Monosialated complex 44.4 1 Bisialated complex 31.9 0Neutral (Z0) 2 0 Monocharged (Z1) 55 1 Bicharged (Z2) 38 2 Tricharged(Z3) 5 3 *Isomeric forms

The quantitative analysis of different glycan structures (Table 6) showsthat the N-glycan forms of factors VII resulting from applicationWO2007/138199 are predominantly of the monosialylated complex type (ataround about 45%). N-glycan forms of the High Mannose/hybrid type arepresent at around 18%.

Example 5: Quantification of the γ-carboxylations

The human factor VIIa has other post-translational modifications, suchas the γ-carboxylation of the N-terminal GLA domain. For the plasmafactor VII, the first ten glutamic acids are all γ-carboxylated(Jurlander B. et al., Recombinant activated factor VII (rFVIIa):characterization, manufacturing, and clinical development. Semin.Thromb. Hemost. 27.4 (2001): 373-84., while the tenth glutamic acid ofNovoSeven® is only partly γ-carboxylated (Thim L. et al., Amino acidsequence and posttranslational modifications of human factor Vlla fromplasma and transfected baby hamster kidney cells. Biochemistry. 27.20(1988): 7785-93).

The study of the γ-carboxylations of factor VII of the invention wasconducted by using the liquid phase chromatography method coupled withmass spectrometry as described below (LC-MS).

The factor VII is reduced and alkylated beforehand and then subject toenzymatic digestion by means of a specific enzyme of the trypsin type.The thereby generated peptides are analyzed by electrospray massspectrometry after separation by reverse phase liquid chromatography ofthe C18 type (Yates, J. R., Ruse, C. I., Nakorchevsky, A., (2009)Proteomics by mass spectrometry: approaches, advances, and applications.Annu Rev Biomed Eng. 11, 49-79.). This approach gives the possibility ofmeasuring the specific mass of the γ-carboxylated N-terminal peptide ofthe factor VII and of inferring therefrom its γ-carboxylation level.

FIG. 2 shows the mass spectrum of the N-terminal peptide [Ala₁-Arg₃₆].The measured average mass is 4.768.0 Da, which is consistent with thetheoretical mass of 4,768.0 Da (reduced and alkylated cysteines) whichcorresponds to 9 γ-carboxylations. The factor VII of the presentinvention therefore has 9 γ-carboxylations. FIG. 2 also contains themass spectrum of the N-terminal peptide [Ala₁-Lys₃₂], which is eluted ina similar retention time. The molecular mass of the peptide is alsoconsistent with the presence of 9 γ-carboxylations on the peptide. Theconclusion may therefore be drawn that the factor VII of the inventionhas 9 γ-carboxylated glutamic residues on the ten potential sites, andthat the glutamic residue located in position 35 relatively to thesequence of the human factor VII (Glu₃₅) is not γ-carboxylated.

Moreover, the peptide [Asp₃₃-Gly₄₇] was isolated by peptide mapping.This peptide contains the amino acid Glu₃₅ which was found in majorityin the non-carboxylated form with a mass of 1,786.9 Da. A minority formof this peptide was discovered as a mass of 1,830.9 Da, corresponding toa modified Glu₃₅. This form is estimated at less than 5%, based on theMS signals. This result confirms the presence of γ-carboxylations on thefirst nine glutamic acids of the factor VII of the invention while thetenth is in majority not γ-carboxylated.

The distribution of the various isoforms of the factor VII of theinvention was also studied by ion exchange chromatography, the procedureof which is described below. The same study was conducted on FVII-rec.

Briefly, the isoforms of the factor VII are separated on a strong anionexchange column (SAX). A sample of factor VII is first loaded on acolumn (Mono Q 5/50 GL, GE Healthcare) equilibrated beforehand to pH 9with a 20 mM Tris buffer. The sample is then eluted by applying anincreasing gradient of an elution buffer of the 20 mM Tris/1M NaCl typeat pH 9.

TABLE 7 Quantification of the isoforms of the factor VII of theinvention and of FVII-rec - Integration of the peaks observed on the «IEX » chromatograms. Quantification of the Retention time isoforms ofthe FVII No. (min) Name of the peak (in %) Factor VII of the invention 126.43 FVII 9GLA 95 2 28.04 FVII 10GLA 5 Total 100 FVII-rec 1 26.58FVII-rec 9GLA 76 2 28.23 FVII-rec 10GLA 24 Total 100 Caption: « FVII9GLA » means factor VII for which the majority isoforms haveγ-carboxylation on nine residues of glutamic acid, « FVII 10GLA » meansfactor VII for which the majority isoforms have γ-carboxylation on 10residues of glutamic acid.

From the results shown in Table 5, the result is that the majorityisoforms of the factor VII of the invention are forms having 9γ-carboxylations (95%). Among the isoforms of FVII-rec, 76% of theisoforms have 9 γ-carboxylations and 24% of the isoforms have 10γ-carboxylations.

Example 6: Study of the Isoelectric Point

The separation of the different isoforms of the factor VII according totheir isoelectric point was achieved by isoelectric focusing «IEF». Theelectrophoretic migration is carried out on a gel Focusgel 3-10 (Serva).

The quality of the factor VII batches is evaluated by analyzing thedifferent isoforms of the product after migration on a gel with a pHgradient 3-10 under native (non-reducing and non-denaturating)conditions, according to the general analysis procedure by IEF on aMultiphor system (GE Healthcare).

The IEF analysis is carried out on Focusgel 3-10 without any SERVA well.The concentration is estimated by measurement of OD at 280 nm (ε=1.36g⁻¹·L·cm⁻¹) after desalting the product on an ultracentrifugationmembrane (cut-off threshold: 10 kDa).

The migration occurs according to the following electric parameters(gradient mode):

Voltage Amperes Power Time Step (V) (mA) (W) (min) 1 375 35 20 29 2 50030 20 1 3 750 30 25 99 4 2000 20 35 1 5 2000 20 35 20

The proteins are revealed by staining with Coomassie Blue CCB-G250.

The determination of the pI of the isoforms is accomplished afterdigitization of the gel and analysis by the Quantity One (Bio-Rad)software package.

The «IEF» profiles of the batch of factor VII of the invention and ofthe FVII-rec are shown in FIG. 4.

The determination of the pI of the bands is accomplished with a standardfor which the pIs are comprised between 5.20 and 8.15. The isoelectricpoint of the factor VII of the invention is comprised between 6.1 and7.3. The profile of the isoforms of the factor VII of the inventionincludes two major bands comprised between 6.6 and 7.0 as well as a moreacid trace at pI 6.1 and a more basic trace at pI 7.3. Both mainisoforms represent about 60% of the totality of the factors VII of thecomposition.

The isoelectric point of the factor VII-rec is comprised between 5.5 and7.5. The profile of the isoforms of the factor VII-rec of the inventionincludes two major bands comprised between 6.4 and 6.8 as well as moreacid traces comprised between 5.5 and 6.4 and more basic traces between6.8 and 7.5. Both main isoforms represent about 45% of the factors VIIof the composition of the FVII-rec.

Therefore, the result of this is that the isoelectric point of thefactor VII of the invention is substantially more homogeneous than theone observed for FVII-rec.

Example 7: Activity of the Factor VII of the Invention

The dosage of the FVII is carried out by an amidolytic method (FVII:am)which measures its biological activity, i.e. the factor VIIa/tissuefactor complex capability of activating the factor X in the presence ofcalcium ions and of phospholipids.

This method is a chromogenic dosage with kinetics in two steps:activation of FX under the action of FVIIa, in a reaction mixturecontaining thromboplastin and calcium; and then enzymatic cleaving of aspecific chromogenic substrate by FXa which releases a chromophore whichmay be quantified by spectrophotometry.

The kinetics readout is accomplished at 405 nm against a referencefilter at 490 nm.

The thereby obtained results are shown in Table 6.

TABLE 8 Dosage of the specific activity of the factor VII of theinvention FVII-Ag: Amidolytic activity Protein level Samples (IU/ml)(mg/ml) FVII of the invention 3307 0.9 (batch 1) FVII of the invention3377 1.1 (batch 2) FVII of the invention 3067 1.0 (batch 3)

The amidolytic activity of factor VII of the invention is comprisedbetween 3,067 IU/ml and 3,377 IU/ml.

The invention claimed is:
 1. A method of making a composition comprisingfactor VII molecules having a substantially homogeneous isoelectricpoint, said method comprising: (a) inserting a DNA sequence comprising agene encoding for factor VII in an embryonic non-human mammal, said genebeing under the transcriptional control of a beta-casein promoter, (b)transferring the embryos obtained in step a) into an oviduct of a femalenon-human mammal so that it develops into an adult mammal, (c) inducinglactation in the adult non-human mammal obtained in step b) of thefemale type or in a female descendant of the non-human mammal whereinthe gene and the promoter are present in its genome, (d) collecting milkof the lactating non-human mammal, and wherein the female non-humanmammal is a rabbit, wherein among all N-glycan forms of the factor VIImolecules, between 70% and 80% of the N-glycan forms are monocharged andbetween 15% and 25% of the N-glycan forms are bicharged, wherein atleast 80% of the factor VII molecules have γcarboxylation on 9 residuesof glutamic acid, and wherein the factor VII originated from transgenicrabbits produced by microinjection of an expression vector comprising abeta-globin insulating sequence from chicken, a control region of goatbeta-casein at 5′, an optimized cDNA sequence for expression in mammalcells coding for human FVII, a non-translated region of beta-casein at3′ (e) purifying factor VII present in the collected milk.
 2. A methodfor formulating a composition of activated factor VII, said methodcomprising mixing a composition of activated factor VII with a buffersolution, adjusting the pH if required, filtering, and then drying ifnecessary to obtain a solid form, wherein the composition of activatedfactor VII comprises factor VII molecules, wherein among all N-glycanforms of the factor VII molecules, between 70% and 80% of the N-glycanforms are monocharged and between 15% and 25% of the N-glycan forms arebicharged, wherein at least 80% of the factor VII molecules haveγcarboxylation on 9 residues of glutamic acid, and wherein the factorVII originated from transgenic rabbits produced by microinjection of anexpression vector comprising a beta-globin insulating sequence fromchicken, a control region of goat beta-casein at 5′, an optimized cDNAsequence for expression in mammal cells coding for human FVII, and anon-translated region of beta-casein at 3′.
 3. The method of claim 2,wherein the steps for mixing the composition of the activated factor VIIwith a buffer solution is applied in gel filtration chromatography. 4.The method of claim 2, further comprising freeze drying the formulatedfactor VII composition.
 5. The method according to claim 2, wherein thebuffer solution comprises one or more of: a) a salt; b) an amino acid, ahydrophilic amino acid salt, or a hydrophobic amino acid salt.
 6. Themethod according to claim 5, wherein the salt is a citrate salt.
 7. Themethod according to claim 6, wherein the citrate salt is trisodiumcitrate.
 8. The method according to claim 5, wherein the hydrophilicamino acid salt is arginine hydrochloride and/or lysine hydrochloride.9. The method according to claim 5, wherein the amino acid is ahydrophobic amino acid.
 10. The method according to claim 9, wherein thehydrophobic amino acid is isoleucine and/or glycine.